Optimizing channel selection for excitation-scanning hyperspectral imaging

摘要

A major benefit of fluorescence microscopy is the now plentiful selection of fluorescent markers. These labelscan be chosen to serve complementary functions, such as tracking labeled subcellular molecules near demarcatedorganelles. However, with the standard 3 or 4 emission channels, multiple label detection is restricted to segregatedregions of the electromagnetic spectrum, as in RGB coloring. Hyperspectral imaging allows the user to discern manyfluorescence labels by their unique spectral properties, provided there is significant differentiation of their emissionspectra. The cost of this technique is often an increase in gain or exposure time to accommodate the signal reductionfrom separating the signal into many discrete excitation or emission channels. Recent advances in hyperspectral imaginghave allowed the acquisition of more signal in a shorter time period by scanning the excitation spectra of fluorophores.Here, we explore the selection of optimal channels for both significant signal separation and sufficient signal detectionusing excitation-scanning hyperspectral imaging.Excitation spectra were obtained using a custom inverted microscope (TE-2000, Nikon Instruments) with a Xe arclamp and thin film tunable filter array (VersaChrome, Semrock, Inc.) Tunable filters had bandwidths between 13 and 17nm. Scans utilized excitation wavelengths between 340 nm and 550 nm. Hyperspectral image stacks were generated andanalyzed using ENVI and custom MATLAB scripts. Among channel consideration criteria were: number of channels,spectral range of scan, spacing of center wavelengths, and acquisition time.